Analysis of a suppressive subtractive hybridization library of Alternaria alternata resistant to 2-propenyl isothiocyanate

Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC.

Toxigenic profile and AFLP variability of Alternaria alternata and Alternaria infectoria occurring on wheat

The objectives of this study were to evaluate the ability to produce alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by A. alternata and A. infectoria strains recovered from wheat kernels obtained from one of the main production area in Argentina; to confirm using AFLPs molecular markers the identify of the isolates up to species level, and to evaluate the intra and inter-specific genetic diversity of these two Alternaria species. Among all the Alternaria strains tested (254), 84% of them were able to produce mycotoxins. The most frequent profile of toxin production found was the co-production of AOH and AME in both species tested. TA was only produced by strains of A. alternata. Amplified fragment polymorphism (AFLPs) analysis was applied to a set of 89 isolates of Alternaria spp (40 were A. infectoria and 49 were A. alternata) in order to confirm the morphological identification. The results showed that AFLPs are powerful diagnostic tool for differentiating between A. alternata and A. infectoria. Indeed, in the current study the outgroup strains, A. tenuissima was consistently classified. Characteristic polymorphic bands separated these two species regardless of the primer combination used. Related to intraspecific variability, A. alternata and A. infectoria isolates evaluated seemed to form and homogeneous group with a high degree of similarity among the isolates within each species. However, there was more scoreable polymorphism within A. alternata than within A. infectoria isolates. There was a concordance between morphological identification and separation up to species level using molecular markers. Clear polymorphism both within and between species showed that AFLP can be used to asses genetic variation in A. alternata and A. infectoria. The most important finding of the present study was the report on AOH and AME production by A. infectoria strains isolated from wheat kernels in Argentina on a semisynthetic media for the first time. Also, specific bands for A. alternata and A. infectoria have been identified; these may be useful for the design of specific PCR primers in order to differentiate these species and to detect them in cereals.